title: Changing nucleotide specificity of the DEAD-box helicase Hera abrogates communication between the Q-motif and the P-loop.
abstract: DEAD-box proteins disrupt or remodel RNA and protein/RNA complexes at the expense of ATP. The catalytic core is composed of two flexibly connected RecA-like domains. The N-terminal domain contains most of the motifs involved in nucleotide binding and serves as a minimalistic model for helicase/nucleotide interactions. A single conserved glutamine in the so-called Q-motif has been suggested as a conformational sensor for the nucleotide state. To reprogram the Thermus thermophilus RNA helicase Hera for use of oxo-ATP instead of ATP and to investigate the sensor function of the Q-motif, we analyzed helicase activity of Hera Q28E. Crystal structures of the Hera N-terminal domain Q28E mutant (TthDEAD_Q28E) in apo- and ligand-bound forms show that Q28E does change specificity from adenine to 8-oxoadenine. However, significant structural changes accompany the Q28E mutation, which prevent the P-loop from adopting its catalytically active conformation and explain the lack of helicase activity of Hera_Q28E with either ATP or 8-oxo-ATP as energy sources. 8-Oxo-adenosine, 8-oxo-AMP, and 8-oxo-ADP weakly bind to TthDEAD_Q28E but in non-canonical modes. These results indicate that the Q-motif not only senses the nucleotide state of the helicase but could also stabilize a catalytically competent conformation of the P-loop and other helicase signature motifs.
authors.affiliation: Institute for Organic and Biomolecular Chemistry, University of Göttingen, Göttingen, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Strohmeier
authors.forename: Ines
authors.initials: I
authors.lastname: Hertel
authors.forename: Ulf
authors.initials: U
authors.lastname: Diederichsen
authors.forename: Markus G
authors.initials: MG
authors.lastname: Rudolph
authors.forename: Dagmar
authors.initials: D
authors.lastname: Klostermeier
mesh_terms: D000225:Adenine / Q000737:chemistry / Q000378:metabolism; D000244:Adenosine Diphosphate / Q000737:chemistry / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000737:chemistry / Q000378:metabolism; D020816:Amino Acid Motifs; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D053487:DEAD-box RNA Helicases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006860:Hydrogen Bonding; D008958:Models, Molecular; D009154:Mutation; D009711:Nucleotides / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology
chemical_list: D009711:Nucleotides; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D053487:DEAD-box RNA Helicases; D000225:Adenine